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7/31/2019 Molecular Control of Brain Development
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Molecular control of braindevelopment
Neuronal Patterning and
Regionalization
2012
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The Spemann Organizer
In 1924, the Ph.D. student Hilde Mangold working in the laboratoryof German embryologist Hans Spemann performed an experimentthat demonstrated that the pattern of development of cells isinfluenced by the activities of other cells
Spemann and Mangold knew that the cells that develop in the
region of the gray crescent migrate into the embryo duringgastrulation and form the notochord (the future backbone; madeofmesoderm).
She cut out a piece of tissue from the gray crescent region of onenewt gastrula and transplanted it into the ventral side of a secondnewt gastrula.
To make it easier to follow the fate of the transplant, she used theembryo of one variety of newt as the donor and a second variety asthe recipient.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Vertebrates.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Vertebrates.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Vertebrates.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Vertebrates.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FrogEmbryology.html7/31/2019 Molecular Control of Brain Development
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The results:
the transplanted tissue developed into a second notochord
neural folds developed above the extra notochord ,these went on to form a second
central nervous system (portions of brain and spinal cord) and eventually a two-
headed tadpole.But the most remarkable finding of all was that the neural folds were built from
recipient cells, not donor cells.
In other words, the transplant had altered the fate of the overlying cells (which
normally would have ended up forming skin [epidermis] on the side of the animal so
that they produced a second head instead!
Spemann and Mangold used the term induction for the ability of one group of cells toinfluence the fate of another. And because of the remarkable inductive power of the
gray crescent cells, they called this region the organizer.
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Organizer Transplant experiment
A region just above the blastopore lip (mesodermal tissue) is
excised & transplanted to ventral side of host.
The host embryo develops a secondary dorsal
axis, first evident by a secondary neural plate.
A section through a host embryo with two dorsal axes:
Secondary dorsal axis contains the same tissues as the
primary dorsal axis, including a nervous system.
Asneural tissue was derived from recipient cells, not
donor cells the transplant had altered the fate of the
overlying cells
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Dorsalization of mesoderm and neural
induction by Spemanns Organizer
during The Organizer experiment
(Spemann and Mangold, 1924) is the
best known experiment in embryology.
It has led to the current view that
development occurs through a cascade
of cell-cell interactions.
If the dorsal lip (the site where
gastrulation starts) of the blastopore is
transplanted to the opposite side of the
embryo, it is able to recruit host cells
organizing them into a secondary
(twinned) body axis containing many
histotypes and complex structures. Spemann referred
to the dorsal lip
as a primary
organizer.
The Organizer of Spemann and Mangold.
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Restriction of Cellular Potency.
The fate of embryonic cells is affected by both the distribution of cytoplasmic
determinants and by cleavage pattern.
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Steps during neural development
Neurogenesis
Compartmentalization
Neural differentiation Neural migration
Axonal guidance
Synaptogenesis
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Neural development in
vertebrate embryo:
Gastrulation
Blastulastage embryo with 3 germ
layers, first signs of invagination of
dorsal blastopore lip
Embryo in midgastrulation,involution of dorsal mesoderm
(organizer tissue).
Gastrula stage embryo:
Embryo at end of gastrulation. The 3
germ layers have arrived at theirfinal destination
Blastula stage through neurulae, highlighting gastrulation and neurulation.
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Organizing Centers:Restricted specialized areas that are crucial for the
induction of area specification
Spemanns organizer (dorsoblastopore lip)
Hensensnode (similar to Spemanns org) Roofplate and notochord become organizers
secondary organizers:
Isthmic organizer (IsO)
Anterior neural ridge (ANR)
Cortical hem
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Default model of neural induction.
Expression of signaling factors:
Bone morphogenic protein (BMP),
a TGF--like polypeptide growthfactor (PGF )expressed in ectodermon ventral side, inducing ectodermto become epidermis.
Organizer on the dorsal side
releases inhibitors of the BMPs:
noggin, chordin, and follistatin,
which diffuse into the ectoderm on
the dorsal side, block the effects of
BMPs, and allow neural tissue to
form.
Balance between agonists and
antagonists!
Importance of inhibition as adevelopmental regulatory
mechanism
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Signaling pathway involving BMPs
Large family of polypeptide growth factors (PGF) related to transforming growthfactor- (TGF-): BMP, activin, and GDF group members.
Heterodimer receptors, with type I & type II subunits, cytoplasmic domains withserine/theronine kinase activity.
Transforming growth factor beta (TGF-beta) and activin bind to receptor complexesthat contain two distantly related transmembrane serine/threonine kinases known asreceptor types I and II. The type II receptors determine ligand binding specificity, andeach interacts with a distinct repertoire of type I receptors.
Dimerization after binding of a TGF--like PGF starts signal transduction pathway:
Activation of cytoplasmic proteins (SMADs), which translocate to nucleus to activateexpression of downstream target genes.
Inhibitory mechanisms regulate signaling:
Extracellular proteins such as chordin, tolloid, and twisted gastrulation interactwith the BMP-like ligands, regulating their diffusion through the extracellularmilieu and their ability to bind receptor
Cell surface proteins such as BAMBI inhibit signaling by binding up BMPs butfailing to transduce a signal.
Inhibitory SMADs poison the signal transduction pathway.
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Signaling transduction pathway
involving BMPs1.Activation of cytoplasmic
proteins (SMADs), whichtranslocate to nucleus toactivate expression ofdownstream target genes.2.Inhibitory mechanismsregulate signaling:
Extracellular proteins such as
chordin, tolloid, and twistedgastrulation interact with theBMP-like ligands, regulatingtheir diffusion through theextracellular milieu and theirability to bind receptorCell surface proteins such as
BAMBI inhibit signaling bybinding up BMPs but failing totransduce a signal.Inhibitory SMADs poison thesignal transduction pathway.
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Neurulation
The neural plate forms after
gastrulation is completed.
The neural tube narrows
along its medial-lateral
Axis. The plate begins to
role into a tube. The
cells at the midline produce
a medial hinge point
(MHP).
As the tube forms andsegregates into the embryo,
neural crest cells emigrate
from the dorsal aspect of the
neural tube.
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Steps during neural development
Neurogenesis
Compartmentalization
Neural differentiation Neural migration
Axonal guidance
Synaptogenesis
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15
Early Neural Patterning:
Establishment of AP Axis
In the early stages of pattern formation, twoperpendicular axes are established
-Anterior/posterior (A/P, head-to-tail) axis
-Dorsal/ventral (D/V, back-to-front) axis
Polarity refers to the acquisition of axialdifferences in developing structures
Position information leads to changes in geneactivity, and thus cells adopt a fateappropriate for their location
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AP polarity of vertebrate CNS
Head organizerbecomesprecordalmesoderm (PME)underneathprechordal plate
Tail organizerbecomesnotochord andsomites,underneathepichordal neuralplate
Head and tailorganizer releasefactors whichcreate a gradient.
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Neural Patterning
A/P polarity and other key organizational features are
first established by gradients ofpositional informationof a gradient of a substance or signal.
Gradient confer positional information as the relativeconcentrations correlated with distance.
In the 3-dimensional system of the embryo, the initial
establishment of A/P polarity is signalled by theorganizer (dorsal lip of the blastopore in amphibians;Hensens node in birds).
During gastrulation, the organizer tissues come to
underlie the neural plate and differentiate into thenotochord.
The chordal mesoderm, which underlies the futuremidbrain, hindbrain, and spinal cord, apparentlysends out distance signals from prechordal
mesoderm.
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The candidate neural inducers, (chordin, noggin, andfollistatin) induce primitive neural tissue that appearsto be forebrain-like; chordin particularly potent.
These 3 proteins antagonize members of TGF- signallingfamily of molecules. This suggests that induction ofanterior neural plate differentiation involves inhibitorsof TGF--like signals that repress neural development.
This would be a ground state, which would be inducedto be more posterior by a 2nd signal: a transformingsignal.
In this case, a type of gradient, a ratio between activating
(noggin) and transforming signals would determinethe A/P polarity along the neuraxis.
Possible candidate posteriorizers (transforming signals)include bFGF and retinoic acid.
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Headorganizer:
BMP
Inhibitors
Cordin and
Noggin,
Wnt
inhibitors
Cerberus,
Dickkopf and
frzb1 to
"anteriorize"
neural tube
Tail organizer:
FGF, WNT, RA &BMP
inhibitors
are posteriorizing
signaling molecules
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The neural tube, shown here for a mouse, is subdivided into four longitudinal
domains: the floor plate, basal plate, alar plate, and roof plate.
Motor neurons are derived from the basal plate.
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Dorsal Ventral pattern: Notochord as organizer
Experiment using Pax gene-Left: During development, the floor plate (red) develops abovethe mesodermal notochord (n) and motor neurons (yellow) differentiate in adjacent
ventrolateral region of the neural tube.
Center: Grafting a donor notochord (n') alongside the folding neural plate results in
formation of an additional floor plate and a third column of motor neurons.
Right: Removing the notochord from beneath the neural plate results in the permanent
absence of both floor plate and motor neurons in the region of the extirpation.Pax6 expression (blue) extends through the ventral region of the cord.
Floor plate cells are induced by sonic hedgehog (SHH) secreted from the
notochord whereas ventral midline cells of the rostral diencephalon (RDVM cells)
appear to be induced by the dual actions of SHH and bone morphogenetic protein
7 (BMP7) from prechordal mesoderm.
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Shh activity in the ventral neural
tube (blue dots) is distributed in a
ventral-high, dorsal-low profilewithin the ventral neural
epithelium.
5 classes of neurons are generated
in response to graded Shh signalling
T.M. Jessell, 2000
Sonic-hedge-hog expression by notochord & floor
plate, control of ventral patterns
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Sonic Hedge hog gene and neuron
Shh acts as a morphogen, forming a gradient in the ventral neural tube, to whichcells differentiate in a concentration- dependent fashion
SHH is a member of the hedgehog family of signalling molecules identified byhomology to the Drosophila hedgehog (HH). SHH is proteolytically cleaved toproduce two secreted proteins , a 19 kDa N-terminal protein (N-SHH) that mediatesall signalling activities in vertebrates and invertebrates and a 25 kDa C-terminalprotein (C-SHH) that possesses protease activity.
N-SHH is responsible for a number of early patterning processes; it is involved inthe control of leftright asymmetry, dorsoventral patterning of the CNS andsomites, patterning of the limb, as well as in some aspects of organogenesis
Sonic hedgehog is a secreted extracellular protein that transmits its signal bybinding to a receptor on the surface of a cell. That binding, in turn, propagates thesignal to the interior of the cell. Once inside, the signal activates a variety of genes
that begin to change a generic neuron into a motor neuron. Signalling by a SHH gradient establishes distinct progenitor domains by regulating
the expression of a set of homeodomain proteins that comprises members of thePax, Nkx, Dbx and Irx families
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(c) Cell types induced byShh vary according to their position
along the anteroposterior axis. Different
colours indicate regional differences in the cell types
differentiating in response to Shh signalling. Dark blue,
ganglionic eminence; pale blue, RDVM cells; brown,
dopaminergic neurons; yellow, serotonergic neurons; green,
motor neurons.
(a) Schematic
representation of the
neural tube and
underlying axial
mesoderm with
regions expressing
Shh indicated in red.Line shows position of
transverse section
shown in (b). T,
telencephalon; D,
diencephalon; M,
midbrain; H,
hindbrain; S.Cord,
spinal
cord; PM, prechordal
mesoderm; NC,notochord.
(b) Transverse
section at the
level of the
spinal cord,
showing
expression of
Shh inthe notochord
and floor plate.
(d) Transverse
section at level
of spinal cord
(indicated inpanel c)
showing ventro-
lateral cell types
(green)
arranged with
bilateral
symmetry
around ventral
midline floorplate cells (red).
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Gradient model for the induction of ventral cell types by Shh.
(a) Distinct ventral cell
types differentiate at
stereotyped
positions in the ventral
neural tube. FP, floor
plate; MN, motor
neurons; V0V3,
classes of ventral
interneurons
generated at spinal
cord levels.
(b) Proposed
gradient of Shh
signal moving
from its sources
of expression in
the ventral neural
tube and
notochord
(c)The
concentration of
Shh required to
induce specific
ventral cell types
in vitro correlates
directly with their
dorso-ventral
position in
vivo.
Patterning
along the
dorso-ventral
axis: a graded
Shh signal
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SHH signalling pathway
At the cell surface, SHH binds with high affinity to patched(Ptc), a 12- transmembrane protein. In mammals, twoisoforms of Ptc are encoded by Ptc1 and Ptc2, althoughPtc1 appears to be active in the CNS .
Binding of SHH to Ptc prevents the normal inhibition ofsmoothened (Smo), a seven-transmembrane protein witha topology reminiscent of G-protein-coupled receptors,which is the signalling component of the SHH-receptorcomplex.
During development of the vertebrate CNS, either inhibitionof Gi proteins or expression of a constitutively activeform of Smo is sufficient to trigger some actions of SHH.
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SHH signalling pathway
Hedgehog-interacting protein (Hip) is a type Itransmembrane protein that attenuates SHH signallingby binding N-SHH with an affinity similar to that of Ptc1
Vitronectin, an extracellular matrix glycoprotein,
enhances SHH activity during motor-neurondifferentiation, also by binding SHH directly.
Within the nucleus of the responding cell, zinc-finger
transcription factors of the Ci/GLI family (GLI13) act atthe last known step of the SHH-signal-transductionpathway , although it is still unclear whether GLI
proteins mediate all aspects of SHH signalling during
vertebrate CNS development .
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Binding of SHH to Ptc
prevents the normal
inhibition of
smoothened (Smo),During development
of the vertebrate CNS,
either inhibition of Gi
proteins or
expression of a
constitutively active
form of Smo is
sufficient to trigger
some actions of SHH.
Within the nucleus of
the responding cell,
zinc-finger
transcription factors
of the Ci/GLI family
(GLI13) act at the lastknown step of the
SHH-signal-
transduction pathway
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(b) Shh initiates the specification of
progenitor cell domains by first exerting a
graded repression of a number of genes which
would otherwise be expressed more widely in
the neural tube. These genes include Pax6, Irx3
and members of the Dbx family
Establishment and maintenance of progenitor cell domains in the ventral neural tube
(a) Progenitor domains corresponding
to the differentiation of specific ventral cell
types (a) are shown on the left-hand side
and indicated with a letter P. Each
domain can be recognised by the
combinatorial pattern of gene expressionshown on the right.
(c) The repression of Pax6 by Shh may
indirectly allow the expression of Nkx2.2 in a
discrete domain adjacent to the floor plate (P3).
A reciprocal repression between these
two genes may then act to refine and maintain the
boundary between the P3 and PMN domains.
Similar mechanisms are believed to
occur at the boundaries between other ventral
progenitor domains.
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Model for ventral neural patterning by SHH.
Left: Graded SHH signaling from
the ventral pole induces
expression of some homeobox
genes (e.g., Nkx2.2, Nkx6.1) and
represses existing expression of
others (e.g. Pax6, Dbx2).
Center: Cross-repressive interactionsbetween pairs of transcription factors
sharpen mutually exclusive expression
domains.
Right: Profiles ofhomeobox gene
expression define
progenitor zones and
control neuronal fate.
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Regulation of DV pattern in the telencephalon by SHH.
Cross section of mouse
telencephalon stage.
SHH produced in the ventral
midline region controls
development of basal ganglia
primordia and medial andlateral ganglionic eminences
(MGE, LGE).
First, ventral SHH induces
medial ganglionic eminences
(MGE) gene expression; SHH
(partly produced by the MGE)induces lateral ganglionic
eminences (LGE )gene
expression later.
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Two Critical Periods of Sonic Hedgehog Signaling Required for
the Specification of Motor Neuron Identity
SHH activity is required for the induction of floor plate differentiation bythe notochord and independently for the induction of motor neurons byboth the notochord and midline neural cells.
Motor neuron generation depends on two critical periods of SHH signaling:
1. an early period during which naive neural plate cells are converted intoventralized progenitors
2. a late period that extends well into S phase of the final progenitor celldivision, during which SHH drives the differentiation of ventralizedprogenitors into motor neurons.
The ambient SHH concentration during the late period determines whetherventralized progenitors differentiate into motor neurons or interneurons,
thus defining the pattern of neuronal cell types generated in the neuraltube.
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b | Dorsal patterning is controlled
by a gradient of bone
morphogenetic proteins (BMPs)
that arises from the dorsal roof
plate, and ventral patterning is
controlled by a gradient of sonic
hedgehog (Shh) that arises fromthe floor plate.
Dorsoventral axis of the spinal cord in quail (bird)
Classes of neurons that can be
identified along the dorsoventral
(DV) axis of the normal embryonic
spinal cord. D, dorsal sensory
neurons; fp, floor plate; mn,
motor neurons; rp, roof plate; V0,
V1, V2, interneurons; V3, ventral
neurons. These classes of neurons
are distinguished by their unique
gene-expression profiles, many of
which are characterized bycombinations of homeobox
transcription factors.
c | The pattern of dorsal and
ventral genes in the retinoic
acid (RA)-depleted quail spinal
cord indicates that there is
increased ventral signalling and
decreased dorsal signalling.
d| The role of RA ingenerating a subset of
motor neurons in the
spinal cord. Retinaldehydedehydrogenase 2 (Raldh2) is expressed
in motor neurons at limb levels (red
circles). A subset of motor neurons
known as lateral motor columnneurons (LMCs) originates close to the
midline of the cord (green circles) and
then migrates through the Raldh2-
expressing motor neurons to
differentiate at the edge of the cord
(arrow). During this journey, these cells
are exposed to RA released by the
motor neurons (red circles), and as a
result, are induced to form LMCs.
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(B) The neural tube in A
differentiates into many different
neuronal types, the major ones
are shown here. Sensory neurons
from the dorsal root ganglia (drg,
purple) enter the dorsal cord and
synapse there. In the ventral
region, the motor neuronsdifferentiate (mn, red). In
between these two are various
types of interneurons with axon
trajectories which connect the
sensory and motor regions (blue
neuron) or connect one side of the
cord to the other (yellow neuron).
VERTEBRATE CNS DEVELOPMENT
Early neural tube with
dorsoventrality :
At the dorsal pole is the roof
plate (rp), a single line of
cells with the nuclei at the
margin, and at the ventral
pole is the floor plate (fp)
where the cells are similarly
arranged.
In the body of the neural
tube, there are many
densely packed neuroblasts,
but towards the ventral
region, there is a swelling
where the neuroblasts are
not so densely packed andthese are the presumptive
motor neurons (mn). (C) Diagram to show the
regionalization of the 6 types of
dorsal neurons (dl1dl6) and the
5 types of ventral neurons (v0v3
+ mn) in the developing neural
tube.
On the left are the gene and
protein markers which are used to
identify the progenitors domains(in the ventricular region close to
the midline) of these different DV
regions. On the right are the gene
and protein markers which are
used to identify the neuronal
types (in the mantle region where
neurons differentiate).
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In the stem zone, FGFs from theunderlying mesoderm (blue) prevent
neural differentiation in the overlying
neural plate (signal 1).
In the transition zone, the notochord
differentiates and starts to express
Shh (yellow, signal 3).
The somites differentiate and start to
express Retinaldehyde dehydrogenase2 , RALDH2 (red) which synthesizes RA
(signal 2). BMPs start to be produced
form the roof plate (green, signal 4).
In the neuronal differentiation and
DV patterning zone, RA antagonizes
FGF and vice versa, RA induces a
specific set of genes in the neuraltube (red arrow), Shh is induced in
the floor plate and spreads dorsally in
a concentration gradient (yellow
arrow), and BMPs spread ventrally in
a concentration gradient (green
arrow).
Summary diagram of the posterior end of the
embryo where DV patterning is taking place.
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Summary of the gene interactions involved in neuronal differentiation in the neural tube. (A) Network
showing the relationship between the inducers of Class I and Class II genes and how they themselves
interact. (B) Later neuronal differentiation of motor neurons involves multiple use of a RA signal and
multiple use of the induction of repressors
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Regionalization of the Nervous System
I. Segmentation
II. Developmental control genes (e.g., Hox), whichencode positional values along A/P axis.A positionalsignaling mechanism activates these genes .
E.G. In birds, At Hensons Node (similar to blastopore ofhigher animals), a strong candidate for this signal isa gradient ofretinoic acid, which regulates thepattern ofHoxgene expression.
Different Hoxgenes at specific locations respond moreor less readily to lower or higher [RA]s, through afamily of receptors, which, bound by RA, becometranscription factors.
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I. Segmentation: Subdivision of the main body axis bysegmentation provides compartments, which allocateprecursor cells into a repeated set of similarmolecules, so that developmental fields can remainsmall, and specialization of cell types and patternscan be generated as local variations on the repetitivetheme.
e.g. Mesoderm = segmented into somites, yielding
muscle groups.The neuraxis is also segmented
In the CNS, segmentation is a mechanism for specifyingpattern during development.
The earliest neurons and neural pathways are laid out instripes, which match a morphological repeat pattern
( a 2-segment repeat pattern, which has similarpatterns of development in even- or odd-numberedsegments).
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Cells are segregated by:
a. Mechanical boundaries (certain extracellular
matrix pattern, such as chondroitin SO4appear at the boundaries during
development (however, only important
during later level.).b. Differential adhesion between cells (occurs
through a 2-segment repeat rule (evens
evens; odds odds), so that adjacent
rhombomeres remain separate.
Compartmental organization of hindbrain into
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Compartmental organization of hindbrain into
rhombomeres in Zebra fish
Genes are expressed in
alternate stripes that
correspond withpresumptive
Rhombomeres.
Restriction of
movement of mitoticPrecursor cells across
interfaces.
The interfaces
betweenRhombomeres acquire
molecular and
Morphological
specialization marked
by distinct boundaries.Julie E. Cooke, Cecilia B. Moens, 2002: Schematic dorsal views of part of thedeveloping vertebrate hindbrain (left side of each panel) and dorsal views of
flat-mounted zebrafish embryo hindbrains at corresponding stages (right side
of each panel). Anterior is to the left; scale bars, 50m
(a) Genes expressed in restricted domains (represented in
red and blue) within the anteroposterior axis of the
hindbrain initially show diffuse boundaries. For example,
krox20 expression (shown on the right as blue signal
following in situ hybridization) shows diffuse boundaries
in presumptive r3 and r5 at bud stage (10 hours post-
fertilization),
(b) Gene expression domain boundaries
progressively sharpen to form straight interfaces.
At 18 somites (18 hours post-fertilization),
domains ofkrox20 expression are sharply
restricted in presumptive r3 and r5.
(c) Gene expression domain boundaries coincide
with structural boundaries; actin accumulation
(shown on the right as red signal after alexa-red-
phalloidin staining) transiently delineates
rhombomere boundaries (white arrowheads)
(d) Mature rhombomere boundary zones are
characterized by large intercellular spaces (white
dots) and concentrations of axons. Different types
of cell differentiate at stereotypical positions withrespect to the boundary (indicated by gradient of
shading across each rhombomere). Expression of
mariposa (shown on the right as blue signal
following in situ hybridization) is localized to
rhombomere boundary zones.
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Mechanism for Hindbrain
Segmentation.
Hindbrain segmentation and the
generation of sharp domains of gene
expression is a two step process.Initial gene expression boundaries in
the hindbrain are diffuse and bi-
directional repulsive signaling
mediated the Eph/ephringene families
leads to a sorting of cells based on
appropriate gene expression.Concomitant with the morphological
formation of rhombomere boundaries,
cells isolated on the wrong side of the
border exhibit plasticity in their gene
expression patterns in response to cell
community signaling effects.Together this leads to the formation
of the sharp molecular and cellular
boundaries that are characteristic of
vertebrate hindbrain development.
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Stages in the compartmental organization of rhombomeres
Genes such as Krox20 and EphA4
(blue) and ephrin-B2 (pink) are
expressed in alternate, fuzzy-
edged stripes (left). Subsequently,
restriction to the movement of
mitotic precursor cells occurs at
the interfaces between newlyformed rhombomeres, which are
now sharply defined, and marked
by increased intercellular spaces.
(right)
Sharpening of boundaries and cell
lineage restriction occur through
the interaction ofEph and ephrin
molecules.
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Neurons and synapseOnce a neuron acquires its individual identity and stops dividing, it extends its
axon with an enlarged tip known as a growth cone.The growth cone is specialized for moving through tissue, using its skills to
select a favourable path. As it does so, it plays out the axon behind it.
Once its target has been reached the growth cone loses its power
of movement and forms a synapse.
Axonal guidance is a supreme navigational feat, accurate over short and
longdistances. It is also a very single-minded process for not
only is the target cell selected with high precision but, to get
there, the growth cone may have to cross over other growth
cones heading for different places. Along the path, guidance
cues that attract (+) or repel (-) the growth cones helpthem find their way, although the molecular mechanisms
responsible for regulating the expression of these cues
remain poorly understood.
Pattern Generation does not Involve only the
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Pattern Generation does not Involve only the
Migration of Cells themselves, but also the
Axons of Cells
Extension or travel of a neuronal axon to a
given area and making specific connections
Appears to involve three steps:pathway selection axons travel to specific region of embryotarget selection recognize and bind a set of cells
address selection refine binding to one or a subset of initial
target
(first two dont depend on neuronal activity)Role of the substrate in directing the pathway of axons has been experimentally
shown as neuronal growth cones prefer to migrate over adhesive surfaces coated
with laminin
Some substrates cause repulsion of axons e.g. ephrin or semiphorin proteins. But all
axons may not be repulsed by these molecules. Some may be attracted.
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The structure of the growth cone is fundamental to its function. The leading edge consists of dynamic, finger-like filopodia that
explore the road ahead, separated by sheets of membrane between the filopodia called lamellipodia-like veils (see the figure).
The cytoskeletal elements in the growth cone underlie its shape, and the growth cone can be separated into three domains based on
cytoskeletal distribution. The peripheral (P) domain contains long, bundled actin filaments (F-actin bundles), which form the filopodia,
as well as mesh-like branched F-actin networks, which give structure to lamellipodia-like veils. Additionally, individual dynamic
'pioneer' microtubules (MTs) explore this region, usually along F-actin bundles. The central (C) domain encloses stable, bundled MTs
that enter the growth cone from the axon shaft, in addition to numerous organelles, vesicles and central actin bundles. Finally, thetransition (T) zone sits at the interface between the P and C domains, where actomyosin contractile structures (termed actin arcs) lie
perpendicular to F-actin bundles and form a hemicircumferential ring. The dynamics of these cytoskeletal components determine
growth cone shape and movement on its journey during development.
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Axon growth, requires (A) the supply of building blocks as well as (B) cycling filaments in thegrowth cone (C) connections between growth cone filaments and the growth substrate
Goldberg J L Genes Dev. 2003;17:941-958
2003 by Cold Spring Harbor Laboratory Press
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The growth cone-Extracellular guidance molecules bind surface receptors on the growth cone.
In turn, these receptors activate signalling cascades that ultimately influence growth cone
cytoskeletal components controlling morphology and motility. Signalling cascades are known
to affect the actin cytoskeleton. It is not clear whether there are also direct influences on
growth cone microtubule assembly during axon guidance.
.
Oster S F , Sretavan D W Br J Ophthalmol 2003;87:639-645
2003 by BMJ Publishing Group Ltd.
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S G ti
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Synapse Generatione.g. formation of a synapse at a neuromuscular junction
a)growth cone approaches muscle cell
b)axon forms unspecified contact on cell surface. Agrin from neuron
causes clustering of Ach receptors
c)neurotransmitter vescicles enter terminal and extracellular matrix
connects the two
d)other axons converge
f)all but one axon eliminated, axon branches, folds form in muscle cell
membrane, Schwann cell covers axon.
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Genetic Control of Dendrite Development
Dendrite arborization patterns are critical determinants of neural circuit
formation and influence the type of synaptic or sensory inputs a neuron isable to receive.
Relatively little is known about the molecular mechanisms that control
dendrite development.
1. Regulation of dendritic field size andcomplexity by transcription factors:
In some cases, the "dendritic fate" of a particular neuron might be specifiedby a single transcription factor. For example, in the Drosophila PNS, the zinc
fingercontaining protein Hamlet functions as a binary switch between the
elaborate multiple-dendrite morphology of the da neuron and the single,
unbranched dendrite morphology of the external sensory (es) neuron.
In most cases, however, the dendritic fate is determined by the combinedaction of multiple transcription factors.
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Genetic Control of Dendrite Development in
Drosophila
Drosophila da (dendritic arborization) neurons fall into four distinctmorphological classes (IIV).
The selector gene Cut is expressed at different levels in the da neurons.Neurons with small and simple dendritic arbors either do not express Cut(class I neurons) or express low levels of Cut (class II). Neurons with morecomplex dendritic branching patterns and larger dendritic fields (classes III
and IV) express higher levels of Cut. Cut levels are a critical determinant ofda neuron class-specific dendritic morphologies.
In contrast to Cut, Spineless (Ss), the homolog of the mammalian dioxinreceptor, is expressed at similar levels in all da neurons. Studies of theepistatic relationship between Cut and Ss indicate that these transcriptionfactors are likely acting in independent pathways to regulate
morphogenesis of da neuron dendrites. More than 70 transcription factors regulate dendritic arbor development
of class I neurons in fly, suggest that complicated networks oftranscriptional regulators likely regulate type-specific dendritearborization patterns.
G ti C t l f D d it
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Genetic Control of Dendrite
Development in Drosophila
2. The molecular mechanism for dendritic self-avoidance and tiling. The dendritesof each da neuron show self-avoidance and tend to spread out without crossingover. Class III and class IV da neurons show tiling; i.e., there is little overlapbetween the dendritic fields of adjacent neurons of the same class because theirdendrites show homotypic repulsion.
Dscam (Down syndrome cell adhesion molecule), is needed for self-avoidance and
contribute to the spreading of dendrites. Without Dscam, the dendrites of each daneuron bundle together or cross over. For the dendritic fields of different neuronsto coexist in the same space, they need to express different Dscam isoforms.
In contrast, tiling requires some cell surface recognition molecules other thanDscam to mediate the homotypic repulsion. The evolutionarily conserved proteinkinase Tricornered (Trc) and the putative adapter protein Furry (Fry) have beenidentified as important components of the intracellular signaling cascade involved
in tiling. 3. Dendrite-specific developmental regulators are a group ofdar(dendritic
arborization reduction) genes. Mutations of any of the dargenes lead to defectivedendritic arbors but normal axonal projections. There may be a total of about 20dargenes in Drosophila.
G ti C t l f D d it
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Genetic Control of Dendrite
Development in Drosophila4. The maintenance of dendritic fields by specific mechanisms.The tumor-suppressor
Warts (Wts), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila(the other being Trc), and the Polycomb group of genes are required for themaintenance of class IV da dendrites. Loss-of-function mutants of any of thosegenes cause a progressive defect in the maintenance of dendritic tiling, resulting inlarge gaps in the receptive field
5. The remodeling of dendritic fields. Drosophila class IV da neurons undergo
dramatic remodeling during metamorphosis. Early in the pupal stage, thoseneurons prune all their dendrites. Later each neuron grows a completely newdendrite for adult function. While the dendrites are being remodeled, the axonsstay largely intact.
Extension of Drosophila Work to Dendrite Development of MammalianCentral Neurons:The great majority of the genes found to affectDrosophila dendrite development have a mammalian homolog(s). Inseveral cases those homologs (for example, Dasm1, Dar3/Sar1) havesimilar function in regulating dendrite development in the mammaliancentral nervous system.
l l l
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II. Developmental Control Genes.
These genes, which encode txn factors, or signaling molecules, areexpressed in a spatially variable manner.
The Hoxgenes (homeobox family) have a clustered chromosomal
organization. The relative position of the gene reflects theexpression along the A/P axis. This expression of the Hoxgeneconfers positional value and regional identity.
The Hox genes are a set of transcription factor genes that exhibit anunusual property: These are genes that specify segment identitywhether a segment of the embryo will form part of the head,thorax, or abdomen, for instanceand they are all clusteredtogether in one (usually) tidy spot. Within that cluster, there is evenfurther evidence of order.
Unlike most genes, however, the order of Hox genes in the genomeactually holds meaning. Hox code represents is a somewhat digital
mechanism for regulating axial patterning. By mixing and matchingcombinations of the expression of a small number of Hox genes,organisms generate a greater range of morphological possibilities
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Hox genes
E.g. Hox Genes in Drosphila :There are eight Hox genes in a row,and the genes' order within that row reflects their order ofexpression in the fly body. The gene found on the left or 3' end ofthe DNA strand, denoted lab (labial), is expressed in the head; onthe other hand, the gene at the right end of the DNA strand, Abd-B(Abdominal-B), is expressed at the end of the fly's abdomen.
Knocking out individual Hox genes in Drosophila causes homeotictransformationsin other words, one body part develops intoanother. A famous example is the Antennapedia mutant, in whichlegs develop on the fly's head instead of antennae.
The Hox genes are early actors in the cascade of interactions thatenable the development of morphologically distinct regions in asegmented animal.e.g. the activation of a Hox gene from the 3' endis one of the earliest triggers that lead the segment to develop intopart of the head.
Genomic Organization of the Hox Gene Cluster
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g
A schematic of the Hox gene clusters (not to scale) in the genomes of D. melanogaster and M.
musculus. Genes are colored to differentiate between Hox family members, and genes that are
orthologous between clusters and species are labeled in the same color. Genes are shown in the
order in which they are found on the chromosomes but, for clarity, some non-Hox genes that are
located within the clusters in the fly genome have been excluded. The positions of three non-Hoxhomeodomain genes, zen, bcd and ftz, are shown in the fly Hox cluster (grey boxes).
Gene abbreviations: lab, labial; pb, proboscipedia; zen, zerknullt; bcd, bicoid; Dfd, Deformed;
Scr, Sex combs reduced; ftz, fushi tarazu; Antp, Antennapedia; Ubx, Ultrabithorax; abd-A,
abdominal-A; Abd-B, Abdominal-B.
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5 3Genes respondmore rapidly at
lower [RA]s
Genes respondless rapidly; require
higher [RA]s
Posterior CNS Anterior CNS
Change in Hoxgene expression
change in morphology along the A/P axisThe signaling mechanism for expression of these genes is a gradient of
Retinoic acid (RA). The RA signal regulates the pattern ofHoxexpression.
There is a direct correspondence between the location of the Hoxgene in its
cluster and its responsiveness to RA.
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Patterning of the brain and spinal cord through
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Patterning of the brain and spinal cord through
compartmentalization:
Regional patterning: Forebrain (FB), Midbrain (MB), Hindbrain (HB) and Spinal cord (SC).
Graded Wnt signaling functions along the entire length of the neuraxis inducing
progressively more posterior neural fates.
Hox genes play important roles in establishing regional cell identity. This is achieved via
opposing gradients ofRA and FGF signaling.
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Regional specification in the developing brain
Three-vesicle state Five-vesicle state
Rh b th l t bdi i i titi th hi db i ith li
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Rhombomeres the clearest subdivision partition the hindbrain neuroepithelium e.g.
CHICK.
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Hox gene expression domains in the CNS
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Hox gene expression domains in the CNS
Nested domains of homeotic genes along the AP axis of the Drosophila and mouse CNS.
Hoxgenes specify a positional value along the AP axis, which is interpreted differently in fly
and mouse in terms of downstream gene activation, resulting in neural structure;
Hirth et al., (1998).
SUMMARY OF MOLECULAR CONTROL OF NERVOUS
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SUMMARY OF MOLECULAR CONTROL OF NERVOUS
DEVELOPMENT
The organizer has three main properties:
1) it induces neural tissue on the overlying
ectoderm,
2) imparts more dorsal characteristics to themesoderm of the marginal zone (i.e.,
dorsalizes mesoderm), leading to the
formation of somites and trunk muscles,
3) it induces a secondary gut (dorsalization of
the endoderm).
The organizer does NOT induce the centralSUMMARY
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nervous system but, instead,
it prevents signals originating from the
ventral side of the blastula from inducing skin
(epidermis) there.
Cells on the ventral side of the blastula
secrete a variety of proteins such as bone
morphogenetic protein-4 (BMP-4)
These induce the ectoderm above to become
epidermis.
If their action is blocked, the ectodermal cells
are allowed to follow their default pathway,
which is to become nerve tissue of the brainand spinal cord.
The Spemann organizer blocks the action of
BMP-4 by secreting molecules of the proteins
chordin and noggin
Both of these physically bind to BMP-4
molecules in the extracellular space and thus
prevent BMP-4 from binding to receptors on
the surface of the overlying ectoderm cells.
This allows the ectodermal cells to follow
their intrinsic path to forming neural folds
and, eventually, the brain and spinal cord.
SUMMARY
SUMMARY
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SUMMARY
Organizers ORGANIZERS IN XENOPUS Protein synthesis by the cells of the organizer
requires transcription of the relevant genes(e.g., chordin).
Expression of organizer genes depends firston Wnt transcription factors. Theirmessenger RNAs were deposited by themother in the vegetal pole of the egg. After
fertilization and formation of the graycrescent, they migrated into the graycrescent region (destined to become theorganizer) where they were translated intoWnt protein.
Wnt protein accumulation on the dorsal sideof the embryo unleashes the activity ofNodal a member of the TGF- family.Nodal induces these dorsal cells to begin
expressing the proteins of Spemann'sorganizer.
ORGANIZERS IN DROSOPHILA Proteins similar in structure to the bone
morphogenetic proteins and also to chordinare found in Drosophila.
The role ofBMP-4 is taken by a relatedprotein encoded by the decapentaplegicgene (dpp).
The role ofchordin is taken by a related
protein called SOGencoded by the genecalled short gastrulation.
In Drosophila, DPP is produced in the dorsalregion of the embryo and SOG is producedin the ventral region.
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Difference in Drosophila and Humans
The actions of SOG on overlying cells are the same asin Xenopus; that is, the SOG protein prevents the DPPprotein from blocking the formation of the centralnervous system. The result in Drosophila is that its
central nervous system forms on the ventral side of theembryo, not on the dorsal! One of the distinguishingtraits of all arthropods (insects, crustaceans, arachnids)as well as many other invertebrates, such as theannelid worms, is a ventral nerve cord.
Chordates, including all vertebrates, have a dorsal(spinal) nerve cord.
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Human nervous system develpment
Cell differentiation begins with the emergence of the cells in thethree primordial layers of the gastrula: the ectoderm (outer layer),the mesoderm (middle layer) and the endoderm (inner layer).
The progenitor cells for all the neurons and glial cells of the centralnervous system begin as a further differentiation of ectoderm cellsinto a layer known as the neural plate.
Neural plate formation is induced by chemical signals from themesoderm (evidently peptides with molecular weight less than1,000). The neural plate folds and differentiates into neural crestcells and a neural tube. The neural crest cells become theperipheral nervous system, whereas the neural tube becomes the
central nervous system. Cells in both structures differentiate into glial cells of various types
as well as into immature neurons which migrate, grow axons &dendrites, form synapses and mature.
Neurulation
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Neurulation
The neural plate forms after
gastrulation is completed.
The neural tube narrows
along its medial-lateral
Axis. The plate begins torole into a tube. The
cells at the midline produce
a medial hinge point
(MHP).
As the tube forms andsegregates into the embryo,
neural crest cells emigrate
from the dorsal aspect of the
neural tube.
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Neurulation Genes give dorsalventral position information.
Sonic hedgehogis an example of a dorsalventralgene that is expressed in the notochord andinduces cells in the overlying neural tube tobecome ventral spinal cord cells.
Another family of homeobox genes, the Pax genes,are important in nervous system and somitedevelopment. Pax3 is expressed in neural tubecells that will become dorsal spinal cord cells.
Pax3 and sonic hedgehog interact to determinedorsalventral differentiation of the spinal cord.
Neurulation.
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(A) During the first phase of
neurulation, nave
uncommitted ectoderm is
induced to form neural plate
tissue via BMP inhibitory
signals (noggin, chordin,
follistatin) secreted from the
underlying mesoderm. (B)
During the second phase of
neurulation the two halves
of the open neural plate
begin to curl up to form a
hollow neural tube. During
this time neural crest cells
(ncc), which express Snail
are induced at the neuralplate border and begin to
migrate in response to Wnt6
and Bmp expression in the
surface ectoderm and dorsal
neural tube respectively.
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Th f d i f h i l d
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The foundation for the anatomical and
functional complexity of the vertebrate
central nervous system is laid during
embryogenesis.
After Spemann's organizer and itsderivatives have endowed the neural plate
with a coarse pattern along its
anteroposterior and mediolateral axes, this
basis is progressively refined by the
activity ofsecondary organizers within the
neuroepithelium that function by releasing
diffusible signaling factors.Dorsoventral patterning is mediated by
two organizer regions that extend along
the dorsal and ventral midlines of the
entire neuraxis, whereas anteroposterior
patterning is controlled by several discrete
organizers.
Organizer signals come from a surprisingly
limited set of signaling factor families,
indicating that the competence of target
cells to respond to those signals plays an
important part in neural patterning.
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Patterning
The underlying principle of patterning is that cells get to know their position relative tothe principal axes of the nervous system - front to back and top to bottom. In effect,
each cell measures its position with respect to these orthogonal coordinates much as
a map-reader figures out his or her position by measuring distance from defined
points.
The way this works at the molecular level is that the embryo sets up a number of
localised polarizing regions in the neural tube that secrete signal molecules. In each
case, the molecule diffuses away from its source to form a gradient of concentration
with distance.
An example of this position-sensing mechanism is the top to bottom (dorsoventral) axis of
the spinal cord. The bottom part of the neural tube expresses a secreted protein -
Sonic hedgehog.Sonic hedgehog diffuses away from the floor plate and affects cells on the dorsoventral
axis according to their distance from the floor plate. When close, Sonic hedgehog
induces the expression of a gene that makes a particular type of interneuron.
Further away, the now lower concentration of Sonic hedgehog induces expression of
another gene making motor neurons.
b
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Hox in vertebrates
Vertebrates, including mice, have Hox genes that are homologous to thoseof the fly, and these genes are clustered in discrete locations with a 3'-to-5' order reflecting an anterior to posterior order of expression.
There are several differences between the mouse and fly Hox genes-
1. there are more Hox genes on the 5' side of the mouse segment; thesecorrespond to expression in the tail, and flies do not have anything
homologous to the chordate tail.2. in the mouse, there are four banks of Hox genes: HoxA, HoxB, HoxC,
and HoxD. Vertebrates have these parallel, overlapping sets of Hoxgenes, which suggest that morphology could be a product of acombinatorial expression of the genes in the four Hox clusters. Thismeans that there could be a Hox code, in which identity can be defined
with more gradations by mixing up the bounds of expression of each ofthe genes.
3. Hox genes are crucial in the orchestration of organized growth inorganisms ranging from plants to humans.
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Differentiation on the anterior
posterior axis is controlled byhomeotic genes.
Four families of genes, called
homeobox or Hox genes, control
differentiation along the body
axis in mice.
Each family consists of 10 genesand resides on a different
chromosome.
Temporal and spatial expression
of these genes follows the same
pattern as their linear order on
their chromosomes.
HOX IN VERTEBRATES
Figure 20.17 Hox Genes Control Body Segmentation (Part 1)
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Figure 20.17 Hox Genes Control Body Segmentation (Part 2)
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Patterns of HOX gene expression in the hindbrain.
HOX genes are expressed in overlapping patterns ending at specific rhombomere
boundaries. Genes at the 3' end of a cluster have the most anterior boundaries, and
paralogous genes have identical expression domains. These genes confer positional value
along the anterior-posterior axis of the hindbrain, determine the identity of the
rhombomeres, and specify their derivatives.
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5 3Genes respondmore rapidly atlower [RA]s
Genes respondless rapidly; require
higher [RA]s
Posterior CNS Anterior CNS
Change in Hoxgene expression
change in morphology along the A/P axisThe signaling mechanism for expression of these genes is a gradient of
Retinoic acid (RA).
The RA signal regulates the pattern ofHoxexpression.
There is a direct correspondence between the location of the Hoxgene in its
cluster and its responsiveness to RA.
Hox genes affect neuronal migration and the
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Hox genes affect neuronal migration and the
development of the mammalian brain
E.g . Hoxa2, controls the pontine neurons' responsiveness tochemicals that attract and repel them, thus telling them where togo in the brain.
The Hoxa2 gene controls expression of the receptor, Robo. Robo isbound to the chemical, Slit, which prevents migrating neurons fromresponding to chemoattractants.
If Hoxa2 is absent , pontine neurons become insensitive to Slitsignaling: the neurons ignore the repellant signal and headprematurely toward the chemoattractant, guiding them into thewrong part of the brain. Thus the pontine neurons go to the bottomof the brainstem instead of going to the cerebellum. The absence ofSlit or Robo causes the same type of abnormal migrations causedby the absence of Hoxa2--further evidence that all three areintegral to the same system.
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V t b t d l t
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Vertebrate development
Although a high degree of precision in both thespatial arrangement of neurons and theirconnectivity is achieved from the outset, thewiring of some parts of the nervous system islater subject to activity-dependentrefinement, such as the pruning of axons andthe death of neurons. These losses may
appear wasteful, but it is not always possibleor desirable to make a complete and perfectbrain by construction alone.
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R f
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References
Journals
Mart E, Bovolenta P Sonic hedgehog in CNS
development: one signal, multiple outputs.
Trends Neurosci. 2002 Feb;25(2):89-96.
Books
Gilbert S.F. Developmental biology
http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Bovolenta%20P[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Bovolenta%20P[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Bovolenta%20P[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Bovolenta%20P[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561